Engineered T cell receptors and immune therapy using the same

ABSTRACT

The present invention pertains to antigen recognizing constructs against COL6A3 antigens. The invention in particular provides novel engineered T cell receptor (TCR) based molecules which are selective and specific for the tumor expressing antigen COL6A3. The TCR of the invention, and COL6A3 antigen binding fragments derived therefrom, are of use for the diagnosis, treatment and prevention of COL6A3 expressing cancerous diseases. Further provided are nucleic acids encoding the antigen recognizing constructs of the invention, vectors comprising these nucleic acids, recombinant cells expressing the antigen recognizing constructs and pharmaceutical compositions comprising the compounds of the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/180,980, filed 5 Nov. 2018, which claims the benefit of U.S.Provisional Application Ser. No. 62/582,202, filed 6 Nov. 2017, andGerman Patent Application No. 10 2017 125 888.4, filed 6 Nov. 2017, thecontent of each of these applications is herein incorporated byreference in their entirety. This application is related toPCT/EP2018/080176, filed 5 Nov. 2018, the content of which isincorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.TXT)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_3000058-012002_ST25.txt” createdon 26 Dec. 2019, and 66,603 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention pertains to antigen recognizing constructs againstCOL6A3 antigens. The invention in particular provides novel engineered Tcell receptor (TCR) based molecules which are selective and specific forthe tumor expressing antigen COL6A3. The TCR of the invention, andCOL6A3 antigen binding fragments derived therefrom, are of use for thediagnosis, treatment and prevention of COL6A3 expressing cancerousdiseases. Further provided are nucleic acids encoding the antigenrecognizing constructs of the invention, vectors comprising thesenucleic acids, recombinant cells expressing the antigen recognizingconstructs and pharmaceutical compositions comprising the compounds ofthe invention.

BACKGROUND OF THE INVENTION

The collagens are a superfamily of proteins that play a role inmaintaining the integrity of various tissues. Collagens areextracellular matrix proteins and have a triple-helical domain as theircommon structural element. Collagen VI is a major structural componentof micro fibrils. The basic structural unit of collagen VI is aheterotrimer of the alpha 1(VI), alpha 2(VI), and alpha 3(VI) collagenchains. The alpha 1(VI) and alpha 2(VI) chains are encoded by the COL6A1and COL6A2 genes, respectively. The protein encoded by the COL6A3 geneis the alpha 3 subunit of type VI collagen (alpha 3(VI) collagen chain)(Bertini et al., 2002 Eur. J. Paediatr. Neurol 6:193-8). COL6A3's geneexpression was previously shown to be associated with the progression ofbreast cancer and was elevated in colon cancer (Smith M J, et al.“Analysis of differential gene expression in colorectal cancer andstroma using fluorescence-activated cell sorting purification” Britishjournal of cancer. 2009; 100:1452-1464; Tilman G et al “Human periostingene expression in normal tissues, tumors and melanoma: evidences forperiostin production by both stromal and melanoma cells” Mol Cancer.2007; 6:80) and as a prognosis marker of colorectal carcinoma (Qiao J etal. “Stroma derived COL6A3 is a potential prognosis marker of colorectalcarcinoma revealed by quantitative proteomics” Oncotarget. 2015 Oct. 6;6(30): 29929-29946). COL6A3 gene locates 2q37 in the human genome andcontains 44 exons. The COL6A3 protein has 3177 amino acids and contains12 Von Willebrand factor type A (vWA) domains, one fibronectin type 3domain and one BPTI/Kunitz family of serine protease inhibitors (KU)domain.

Targets for T-cell based immunotherapy represent peptide epitopesderived from tumor-associated or tumor-specific proteins, which arepresented by molecules of the major histocompatibility complex (MHC).These tumor associated antigens (TAAs) can be peptides derived from allprotein classes, such as enzymes, receptors, transcription factors, etc.which are expressed and, as compared to unaltered cells of the sameorigin, usually up-regulated in cells of the respective tumor.

Specific elements of the cellular immune response are capable ofselectively recognizing and destroying tumor cells. The isolation oftumor antigen-specific T cells from tumor-infiltrating cell populationsor from peripheral blood suggests that such cells play an important rolein natural immune defense against cancer. CD8-positive T cells inparticular, which recognize class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10amino acid residues derived from proteins or defective ribosomalproducts (DRiPs) located in the cytosol, play an important role in thisresponse. The MHC-molecules of the human are also designated as humanleukocyte-antigens (HLA).

A TCR is a heterodimeric cell surface protein of the immunoglobulinsuper-family, which is associated with invariant proteins of the CD3complex involved in mediating signal transduction. TCRs exist as αβ andγδ heterodimers, which are structurally similar but have quite distinctanatomical locations and probably functions. The extracellular portionof native heterodimeric αβTCR consists of two polypeptide chains, eachof which has a membrane-proximal constant domain, and a membrane-distalvariable domain. Each of the constant and variable domains includes anintra-chain disulfide bond. The variable domains contain the highlypolymorphic loops analogous to the complementarity determining regions(CDRs) of antibodies. The use of TCR gene therapy overcomes a number ofcurrent hurdles. It allows equipping patients' own T cells with desiredspecificities and generation of sufficient numbers of T cells in a shortperiod of time, avoiding their exhaustion. The TCR will be transducedinto central memory T cells or T cells with stem cell characteristics,which may ensure better persistence and function upon transfer.TCR-engineered T cells will be infused into cancer patients renderedlymphopenic by chemotherapy or irradiation, allowing efficientengraftment but inhibiting immune suppression.

While advances have been made in the development of molecular-targetingdrugs for cancer therapy, there remains a need in the art to develop newanti-cancer agents that specifically target molecules highly specific tocancer cells. The present description addresses that need by providingnovel engineered COL6A3 TCRs, respective recombinant TCR constructs,nucleic acids, vectors and host cells that specifically bind COL6A3epitope(s) as disclosed; and methods of using such molecules in thetreatment of cancer.

SUMMARY OF THE INVENTION

In a first aspect the object of the invention is solved by an antigenrecognizing construct comprising a first domain comprising threecomplementary determining regions (CDRs) according to SEQ ID NOs: 5(CDRa1), 6 (CDRa2), and 7 (CDRa3), and a second domain comprising threecomplementary determining regions (CDRs) according to SEQ ID NOs: 13(CDRb1), 14 (CDRb2), and 15 (CDRb3), wherein at least one of saidcomplementary determining regions is replaced with at least one sequenceselected from the group of: a) SEQ ID NO: 26 (CDRa1-mut1), and SEQ IDNOs: 37 to 49 (CDRb1-mut1 to CDRb1-mut13), preferably SEQ ID NO: 40, andb) mutated sequences of SEQ ID NO: 26, and SEQ ID NOs: 37 to 49,preferably SEQ ID NO: 40, comprising conservative amino acid exchanges.

In another aspect, CDRa1 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% identity with the amino acid sequence according toSEQ ID NO: 5.

In another aspect, CDRa2 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity with the amino acid sequenceaccording to SEQ ID NO: 6.

In another aspect, CDRa3 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity with the amino acid sequenceaccording to SEQ ID NO: 7.

In another aspect, CDRb1 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity with the amino acid sequenceaccording to SEQ ID NO: 13.

In another aspect, CDRb2 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity with the amino acid sequenceaccording to SEQ ID NO: 14.

In another aspect, CDRb3 of an antigen recognizing construct may have atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, at least about 96%, at least about 97%, at least about98%, or at least about 99% identity with the amino acid sequenceaccording to SEQ ID NO: 15.

In the context of the present invention, the inventors have identifiedimproved engineered versions of the COL6A3 TCR R4P3F9 comprising mutatedCDR1 sequences in the alpha and beta chain(s) that are improvingstability, recognition and selectivity of the parental R4P3F9. Thesematurated TCR variants have been selected in a two-step method, whereinone step selects for stability and the second for affinity of thevariants (see examples). While the inventive TCRs comprise mutated CDR1regions, it is likely that CDR2 and or CDR3 can be mutated as well inorder to increase binding affinity/specificity and/or selectivity andsuch mutated CDRs ideally could be included in the existing constructs.

The affinity maturation identified variants with considerably strongerbinding activity towards HLA-A*02/COL6A3-peptide, while retaining oreven improving specificity. Compared to the parental TCR C-1 (R4P3F9 TCRcomprising wild type CDRs, see Table 5), all variants of the inventionimproved IFN-gamma release with higher levels reached already for lowerpeptide loading concentrations.

Within the variable domain, CDR1 and CDR2 are found in the variable (V)region of a polypeptide chain, and CDR3 includes some of V, all ofdiversity (D) and joining (J) regions. It is assumed that CDR3 is themost variable and is the main CDR responsible for specifically andselectively recognizing an antigen. Surprisingly, for some TCRs CDR1seems also to make contacts to the peptide and thus is also responsiblefor selective recognition. In the present case, without being bound to aspecific theory, the mutated CDR1b seems to interact with position 8 ofthe COL6A3-peptide.

Native alpha-beta heterodimeric TCRs have an alpha chain and a betachain. Each alpha chain comprises variable, joining and constantregions, and the beta chain also usually contains a short diversityregion between the variable and joining regions, but this diversityregion is often considered as part of the joining region. Each variableregion comprises three CDRs (Complementarity Determining Regions)embedded in a framework sequence, one being the hypervariable regionnamed CDR3. There are several types of alpha chain variable (Vα) regionsand several types of beta chain variable (Vβ) regions distinguished bytheir framework, CDR1 and CDR2 sequences, and by a partly defined CDR3sequence. The Vα types are referred to in IMGT nomenclature by a uniqueTRAV number, Vβ types are referred to by a unique TRBV number.

Preferably, an antigen recognizing construct of the invention comprisesthe respective CDR1 to CDR3 of one individual herein disclosedengineered TCR variable region of the invention. Preferred are antigenrecognizing constructs (e.g., αβ and γδ TCRs) of the invention whichcomprise at least one, preferably two, maturated CDR1 sequences.

The CDR-variants as disclosed herein—in particular the CDR1-variants—canbe modified by the substitution of one or more residues at different,possibly selective, sites within the peptide chain, if not otherwisestated. Such substitutions are of a conservative nature where one aminoacid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions”. Preferred conservative substitutions are herein definedas exchanges within one of the following five groups: Group 1—smallaliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro,Gly); Group 2—polar, negatively charged residues and their amides (Asp,Asn, Glu, Gln); Group 3—polar, positively charged residues (His, Arg,Lys); Group 4—large, aliphatic, nonpolar residues (Met, Leu, Ile, Val,Cys); and Group 5—large, aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue.

The term “specificity” or “antigen specificity” or “specific for” agiven antigen, as used herein means that the antigen recognizingconstruct can specifically bind to said antigen, preferably a COL6A3antigen, more preferably with high avidity, when said antigen ispresented by HLA, preferably HLA A2. For example, a TCR, as antigenrecognizing construct, may be considered to have “antigenic specificity”for COL6A3 antigens, if T cells expressing the TCR in response to COL6A3presenting HLA secrete at least about 200 pg/ml or more (e.g., 250 pg/mlor more, 300 pg/ml or more, 400 pg/ml or more, 500 pg/ml or more, 600pg/ml or more, 700 pg/ml or more, 1000 pg ml or more, 2,000 pg/ml ormore, 2,500 pg/ml or more, 5,000 pg/ml or more) of interferon γ (IFN-γ)upon co-culture with HLA A2 target cells pulsed with a low concentrationof a COL6A3 antigen, such as the COL6A3 epitopes and antigens providedherein below (e.g., about 10-11 mol/l, 10-10 mol/l, 10-9 mol/l, 10-8mol/l, 10-7 mol/l, 10-6 mol/l, 10-5 mol/l). Alternatively, oradditionally, a TCR may be considered to have “antigenic specificity”for COL6A3, if T cells expressing the TCR secrete at least twice as muchIFN-γ as the untransduced background level of IFN-γ upon co-culture withtarget cells pulsed with a low concentration of COL6A3 antigens. Such a“specificity” as described above can—for example—be analyzed with anELISA.

In one alternative or additional embodiment of the invention, theantigen recognizing construct is stable, and capable of specificallyand/or selectively binding to a COL6A3 antigen; preferably wherein theCOL6A3 antigen is a protein epitope or peptide having an amino acidsequence shown in SEQ ID NO: 1 or a variant thereof, wherein the variantis an amino acid deletion, addition, insertion or substitution of notmore than three, preferably two and most preferably not more than oneamino acid position.

The term “selectivity” or “selective recognizing/binding” is understoodto refer to the property of an antigen recognizing construct, such as aTCR or antibody, to selectively recognize or bind to preferably only onespecific epitope and preferably shows no or substantially nocross-reactivity to another epitope. Preferably “selectivity” or“selective recognizing/binding” means that the antigen recognizingconstruct (e.g., a TCR) selectively recognizes or binds to preferablyonly one specific epitope and preferably shows no or substantially nocross-reactivity to another epitope, wherein said epitope is unique forone protein, such that the antigen recognizing construct shows no orsubstantially no cross-reactivity to another epitope and anotherprotein.

The antigen recognizing construct according to the invention ispreferably selected from an antibody, or derivative or fragment thereof,a bispecific molecule, or a T cell receptor (TCR), or a derivative orfragment thereof. A derivative or fragment of an antibody or TCR of theinvention shall preferably retain the antigen binding/recognizingability of the parent molecule, in particular its specificity and/orselectivity as explained above.

In an embodiment of the invention, the inventive engineered TCRs areable to recognize COL6A3 antigens in a major histocompatibility complex(MHC) class I-dependent manner. “MHC class I-dependent manner,” as usedherein, means that the TCR elicits an immune response upon binding toCOL6A3 peptide antigens within the context of an MHC class I molecule.The MHC class I molecule can be any MHC class I molecule known in theart, e.g., HLA-A molecules. In a preferred embodiment of the invention,the MHC class I molecule is an HLA-A2 molecule.

The invention provides both single chain antigen recognizing constructand double chain recognizing constructs, as well as other variantmolecules. The invention in particular provides an engineered TCR asantigen recognizing construct, or fragment or derivative thereof. Theengineered TCR preferably is of human origin, which is understood asbeing generated from a human TCR locus and therefore comprising humanTCR sequences. Furthermore, the TCR of the invention is characterized asaffinity maturated TCR, which is capable of specifically and selectivelyrecognizing COL6A3 peptide antigen. Another embodiment of the inventionadditionally or alternatively provides the antigen recognizing constructdescribed above, which induces an immune response, preferably whereinthe immune response is characterized by an increase in interferon (IFN)γ levels.

TCRs of the invention may be provided as single chain α or β, or γ andδ, molecules, or alternatively as double chain constructs composed ofboth the α and β chain, or γ and δ chain.

Most preferably, in some additional embodiments, wherein the disclosurerefers to antigen recognizing constructs comprising any one, two or allof the CDR1 to CDR3 regions of the herein disclosed engineered TCRchains (see table 1). Antigen recognizing constructs may be preferred,which comprise the natural or the engineered CDR sequence having three,two, and preferably only one, modified amino acid residues. A modifiedamino acid residue may be selected from an amino acid insertion,deletion or substitution. Most preferred is that the three, two,preferably one modified amino acid residue is the first or last aminoacid residue of the respective CDR sequence. If the modification is asubstitution then it is preferable in some embodiments that thesubstitution is a conservative amino acid substitution.

The inventive TCRs may further comprise a constant region derived fromany suitable species, such as any mammal, e.g., human, rat, monkey,rabbit, donkey, or mouse. In an embodiment of the invention, theinventive TCRs further comprise a human constant region. In somepreferred embodiments, the constant region of the TCR of the inventionmay be slightly modified, for example, by the introduction ofheterologous sequences, preferably mouse sequences, which may increaseTCR expression and stability. Also, further stabilizing mutations asknown from the state of the art (e.g., DE 10 2016 123 893.7) may beintroduced, such as replacement of unfavorable amino acids in the Vregions and/or the introduction of a disulfide bridge between the TCR Cdomains and the removal of unpaired cysteine.

As used herein, the term “murine” or “human,” when referring to anantigen recognizing construct, or a TCR, or any component of a TCRdescribed herein (e.g., complementarity determining region (CDR),variable region, constant region, α chain, and/or β chain), means a TCR(or component thereof), which is derived from a mouse or a humanunrearranged TCR locus, respectively.

In an embodiment of the invention, chimeric TCR are provided, whereinthe TCR chains comprise sequences from multiple species. Preferably, aTCR of the invention may comprise an α chain comprising a human variableregion of an α chain and, for example, a murine constant region of amurine TCR α chain.

In one embodiment, the TCR of the invention is a human TCR comprisinghuman variable regions according to the above embodiments and humanconstant regions.

The TCR of the invention may be provided as a single chain TCR (scTCR).A scTCR can comprise a polypeptide of a variable region of a first TCRchain (e.g., an alpha chain) and a polypeptide of an entire(full-length) second TCR chain (e.g., a beta chain), or vice versa.Furthermore, the scTCR can optionally comprise one or more linkers whichjoin the two or more polypeptides together. The linker can be, forinstance, a peptide, which joins together two single chains, asdescribed herein. Also provided is such a scTCR of the invention, whichis fused to a human cytokine, such as IL-2, IL-7 or IL-15.

The antigen recognizing construct according to the invention can also beprovided in the form of a multimeric complex, comprising at least twoscTCR molecules, wherein said scTCR molecules are each fused to at leastone biotin moiety, and wherein said scTCRs are interconnected bybiotin-streptavidin interaction to allow the formation of saidmultimeric complex. Similar approaches for the generation of multimericTCR are also possible and included in this disclosure. Also provided aremultimeric complexes of a higher order, comprising more than two scTCRof the invention.

For the purposes of the present invention, a TCR is a moiety having atleast one TCR alpha or gamma and/or TCR beta or delta variable domain.Generally, they comprise both a TCR alpha variable domain and a TCR betavariable domain. They may be αβ heterodimers or may be in single chainformat. For use in adoptive therapy, an αβ heterodimeric TCR may, forexample, be transfected as full length chains having both cytoplasmicand transmembrane domains. If desired, an introduced disulfide bondbetween residues of the respective constant domains may be present.

In a preferred embodiment, the antigen recognizing construct is a humanTCR, or fragment or derivative thereof. A human TCR or fragment orderivative thereof is a TCR, which comprises over 50% of thecorresponding human TCR sequence. Preferably, only a small part of theTCR sequence is of artificial origin or derived from other species. Itis known, however, that chimeric TCRs, e.g., derived from human originwith murine sequences in the constant domains, are advantageous.Particularly preferred are, therefore, TCRs in accordance with thepresent invention, which contains murine sequences in the extracellularpart of their constant domains.

Thus, it is also preferred that the inventive antigen recognizingconstruct is able to recognize its peptide antigen in a human leucocyteantigen (HLA) dependent manner, preferably in an HLA-A02 dependentmanner. The term “HLA dependent manner” in the context of the presentinvention means that the antigen recognizing construct binds to theantigen only in the event that the antigenic peptide is presented bysaid HLA.

The antigen recognizing construct in accordance with the invention inone embodiment preferably induces an immune response, preferably whereinthe immune response is characterized by the increase in interferon (IFN)γ levels.

The term “polypeptide” as used herein includes oligopeptides and refersto a single chain of amino acids connected by one or more peptide bonds.With respect to the inventive polypeptides, the functional portion canbe any portion comprising contiguous amino acids of the TCR (orfunctional variant thereof), of which it is a part, provided that thefunctional portion specifically binds to a COL6A3 antigen, preferably asdisclosed herein in table 1. The term “functional portion” when used inreference to a TCR (or functional variant thereof) refers to any part orfragment of the TCR (or functional variant thereof) of the invention,which part or fragment retains the biological activity of the TCR (orfunctional variant thereof), of which it is a part (the parent TCR orparent functional variant thereof). Functional portions encompass, forexample, those parts of a TCR (or functional variant thereof) thatretain the ability to specifically bind to a COL6A3 antigen (in anHLA-A2-dependent manner), or detect, treat, or prevent cancer, to asimilar extent, the same extent, or to a higher extent, as the parentTCR (or functional variant thereof).

The functional portion can comprise additional amino acids at the aminoor carboxy terminus of the portion, or at both termini, in whichadditional amino acids are not found in the amino acid sequence of theparent TCR or functional variant thereof. Desirably, the additionalamino acids do not interfere with the biological function of thefunctional portion, e.g., specifically binding to COL6A3 antigens;and/or having the ability to detect cancer, treat or prevent cancer,etc. More desirably, the additional amino acids enhance the biologicalactivity, as compared to the biological activity of the parent TCR orfunctional variant thereof.

As already mentioned above, the binding functionality of the TCR of theinvention may be provided in the framework of an antibody. The term“antibody” in its various grammatical forms is used herein to refer toimmunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain anantigen-binding site or a paratope. Such molecules are also referred toas “antigen binding fragments” of immunoglobulin molecules. Theinvention further provides an antibody, or antigen binding portionthereof, which specifically binds to the antigens described herein. Theantibody can be any type of immunoglobulin that is known in the art. Forinstance, the antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG,IgM, etc. The antibody can be monoclonal or polyclonal. The antibody canbe a naturally-occurring antibody, e.g., an antibody isolated and/orpurified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,hamster, human, etc. Alternatively, the antibody can be agenetically-engineered antibody, e.g., a humanized antibody or achimeric antibody. The antibody can be in monomeric or polymeric form.

The invention also provides antigen binding portions of any of theantibodies described herein. The antigen binding portion can be anyportion that has at least one antigen binding site, such as Fab,F(ab′)2, dsFv, scFv, diabodies, and triabodies. A single-chain variableregion fragment (scFv) antibody fragment, which consists of a truncatedFab fragment comprising the variable (V) domain of an antibody heavychain linked to a V domain of a light antibody chain via a syntheticpeptide, can be generated using routine recombinant DNA technologytechniques. Similarly, disulfide-stabilized variable region fragments(dsFv) can be prepared by recombinant DNA technology, antibody fragmentsof the invention, however, are not limited to these exemplary types ofantibody fragments. Also, the antibody, or antigen binding portionthereof, can be modified to comprise a detectable label, such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Kohler andMilstein, Eur. J. Immunol, 5, 51 1-519 (1976), Harlow and Lane (eds.),Antibodies: A Laboratory Manual, CSH Press (1988), and C. A. Janeway etal. (eds.), Immunobiology, 8 Ed., Garland Publishing, New York, N.Y.(2011)). Alternatively, other methods, such as EBV-hybridoma methods(Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), andRoder et al, Methods Enzymol, 121, 140-67 (1986)), and bacteriophagevector expression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266.

Some embodiments of the invention also pertain to TCRs, or functionalfragments and polypeptides thereof, which are soluble TCRs. As usedherein, the term “soluble T-cell receptor” refers to single chain orheterodimeric truncated variants of native TCRs, which comprise at leastthe variable domains of the TCR α-chain and β-chain linked by apolypeptide linker (SEQ ID NOs: 22, 24, 25 and 27). Soluble variants ofTCRs usually lack at least the transmembrane and cytosolic domains ofthe native protein; sometimes preferably such soluble constructs do notcomprise any constant domain sequences. The soluble T-cell receptorconstructs of the invention, in preferred embodiments, compriseconstructs consisting of α- and β-chain variable domain sequences asprovided herein, connected by a suitable linker sequence. The variabledomain sequence (amino acid or nucleic acid) of the soluble TCR α-chainand β-chains may be identical to the corresponding sequences in a nativeTCR or may comprise variants of soluble TCR α-chain and β-chain variabledomain sequences, as compared to the corresponding native TCR sequences.The term “soluble T-cell receptor” as used herein encompasses solubleTCRs with variant or non-variant soluble TCR α-chain and β-chainvariable domain sequences. The variations may be in the framework and/orCDR regions of the soluble TCR α-chain and β-chain variable domainsequences and can include, but are not limited to, amino acid deletion,insertion, substitution mutations as well as changes to the nucleic acidsequence, which do not alter the amino acid sequence. Soluble TCR of theinvention in any case retain or preferentially improve the bindingfunctionality of their parental TCR molecules.

The above problem is further solved by a nucleic acid encoding for anantigen recognizing construct of the invention, or any of theaforementioned protein or polypeptide constructs. The nucleic acidpreferably (a) has a strand encoding for an antigen recognizingconstruct according to the invention; (b) has a strand complementary tothe strand in (a); or (c) has a strand that hybridizes under stringentconditions with a molecule as described in (a) or (b). Stringentconditions are known to the person of skill in the art, specificallyfrom Sambrook et al, “Molecular Cloning”. In addition to that, thenucleic acid optionally has further sequences, which are necessary forexpressing the nucleic acid sequence corresponding to the protein,specifically for expression in a mammalian/human cell. The nucleic acidused can be contained in a vector suitable for allowing expression ofthe nucleic acid sequence corresponding to the polypeptide in a cell.However, the nucleic acids can also be used to transform anantigen-presenting cell, which may not be restricted to classicalantigen-presenting cells, such as dendritic cells, in such a way thatthey themselves produce the corresponding proteins on their cellularsurface.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and can contain a natural, non-natural or altered internucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide.

Preferably, the nucleic acids of the invention are recombinant. As usedherein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication. The nucleic acid can compriseany nucleotide sequence, which encodes any of the TCRs, polypeptides, orproteins, or functional portions or functional variants thereofdescribed herein.

Furthermore, the invention provides a vector comprising a nucleic acidin accordance to the invention as described above. Desirably, the vectoris an expression vector or a recombinant expression vector. The term“recombinant expression vector” refers in context of the presentinvention to a nucleic acid construct that allows for the expression ofan mRNA, protein or polypeptide in a suitable host cell. The recombinantexpression vector of the invention can be any suitable recombinantexpression vector and can be used to transform or transfect any suitablehost. Suitable vectors include those designed for propagation andexpansion or for expression or both, such as plasmids and viruses.Examples of animal expression vectors include pEUK-Cl, pMAM, andpMAMneo. Preferably, the recombinant expression vector is a viralvector, e.g., a retroviral vector. The recombinant expression vectorcomprises regulatory sequences, such as transcription and translationinitiation and termination codons, which are specific to the type ofhost cell (e.g., bacterium, fungus, plant, or animal), into which thevector is to be introduced and in which the expression of the nucleicacid of the invention may be performed. Furthermore, the vector of theinvention may include one or more marker genes, which allow forselection of transformed or transfected hosts. The recombinantexpression vector can comprise a native or normative promoter operablylinked to the nucleotide sequence encoding the constructs of theinvention, or to the nucleotide sequence, which is complementary to orwhich hybridizes to the nucleotide sequence encoding the constructs ofthe invention. The selections of promoters include, e.g., strong, weak,inducible, tissue-specific and developmental-specific promoters. Thepromoter can be a non-viral promoter or a viral promoter. The inventiverecombinant expression vectors can be designed for either transientexpression, for stable expression, or for both. Also, the recombinantexpression vectors can be made for constitutive expression or forinducible expression.

The invention also pertains to a host cell comprising an antigenrecognizing construct in accordance with the invention. Specifically,the host cell of the invention comprises a nucleic acid, or a vector asdescribed herein above. The host cell can be a eukaryotic cell, e.g.,plant, animal, fungi, or algae, or can be a prokaryotic cell, e.g.,bacteria or protozoa. The host cell can be a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell can be an adherent cell or a suspended cell, i.e., a cell thatgrows in suspension. For purposes of producing a recombinant TCR,polypeptide, or protein, the host cell is preferably a mammalian cell,e.g., Chinese Hamster Ovary (CHO) cell. Most preferably, the host cellis a human cell. While the host cell can be of any cell type, canoriginate from any type of tissue, and can be of any developmentalstage, the host cell preferably is a peripheral blood leukocyte (PBL) ora peripheral blood mononuclear cell (PBMC). More preferably, the hostcell is a T cell. The T cell can be any T cell, such as a cultured Tcell, e.g., a primary T cell, or a T cell from a cultured T cell line,e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal,preferably a T cell or T cell precursor from a human patient. Ifobtained from a mammal, the T cell can be obtained from numeroussources, including but not limited to blood, bone marrow, lymph node,the thymus, or other tissues or fluids. T cells can also be enriched foror purified. Preferably, the T cell is a human T cell. More preferably,the T cell is a T cell isolated from a human. The T cell can be any typeof T cell and can be of any developmental stage, including but notlimited to, CD4-positive and/or CD8-positive, CD4-positive helper Tcells, e.g., Th1 and Th2 cells, CD8-positive T cells (e.g., cytotoxic Tcells), tumor infiltrating cells (TILs), memory T cells, naive T cells,and the like. Preferably, the T cell is a CD8-positive T cell or aCD4-positive T cell.

Preferably, the host cell of the invention is a lymphocyte, preferably,a T lymphocyte, such as a CD4-positive or CD8-positive T-cell. The hostcell furthermore preferably is a tumor reactive T cell specific forCOL6A3 expressing tumor cells.

One further aspect of the present invention relates to the hereindisclosed antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell for use in medicine. Theuse in medicine in one preferred embodiment includes the use in thediagnosis, prevention and/or treatment of a tumor disease, such as amalignant or benign tumor disease. The tumor disease is, for example, atumor disease characterized by the expression of COL6A3, in a cancer ortumor cell of said tumor disease.

With respect to the above mentioned medical applications of the antigenrecognizing constructs and other materials derived therefrom, the to betreated and/or diagnosed cancer can be any cancer, including any ofacute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vagina, cancer of the vulva,chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer,esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor,glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynxcancer, liver cancer, lung cancer, malignant mesothelioma, melanoma,multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer ofthe oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer, skin cancer, small intestinecancer, soft tissue cancer, stomach cancer, testicular cancer, thyroidcancer, cancer of the uterus, ureter cancer, and urinary bladder cancer.A preferred cancer is cancer is cancer of the uterine cervix,oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis. Aparticularly preferred cancer is a COL6A3 positive cancer, includinggastrointestinal and gastric cancer.

The constructs, proteins, TCRs, antibodies, polypeptides and nucleicacids of the invention are in particular for use in immune therapy,preferably, in adoptive T cell therapy. The administration of thecompounds of the invention can, for example, involve the infusion of Tcells of the invention into said patient. Preferably, such T cells areautologous T cells of the patient and in vitro transduced with a nucleicacid or antigen recognizing construct of the present invention.

WO 2016/011210 discloses engineered cells for adoptive therapy,including NK cells and T cells, and compositions containing the cells,and methods for their administration to subjects. The cells can containgenetically engineered antigen receptors that specifically bind toantigens, such as chimeric antigen receptors (CARs) and costimulatoryreceptors.

The object of the invention is also solved by a method of manufacturinga COL6A3 specific antigen recognizing construct expressing cell line,comprising

-   a. providing a suitable host cell,-   b. providing a genetic construct comprising a coding sequence    encoding the antigen recognizing construct according to any of    claims 1 to 4,-   c. introducing said genetic construct into said suitable host cell,    and-   d. expressing said genetic construct by said suitable host cell.

The method may further comprise a step of cell surface presentation ofsaid antigen recognizing construct on said suitable host cell.

In other preferred embodiments, the genetic construct is an expressionconstruct comprising a promoter sequence operably linked to said codingsequence.

Preferably, said antigen recognizing construct is of mammalian origin,preferably of human origin. The preferred suitable host cell for use inthe method of the invention is a mammalian cell, such as a human cell,in particular a human T lymphocyte. T cells for use in the invention aredescribed in detail herein above.

Also encompassed by the invention are embodiments, wherein said antigenrecognizing construct is a modified TCR, wherein said modification isthe addition of functional domains, such as a label or a therapeuticallyactive substance. Furthermore, encompassed are TCR having alternativedomains, such as an alternative membrane anchor domain instead of theendogenous transmembrane region.

Desirably, the transfection system for introducing the genetic constructinto said suitable host cell is a retroviral vector system. Such systemsare well known to the skilled artisan.

Also comprised by the present invention is in one embodiment theadditional method step of isolation and purification of the antigenrecognizing construct from the cell and, optionally, the reconstitutionof the translated antigen recognizing construct-fragments in a T-cell.

In an alternative aspect of the invention a T-cell is provided obtainedor obtainable by a method for the production of a T cell receptor (TCR),which is specific for tumorous cells and has high avidity as describedherein above. Such a T cell is depending on the host cell used in themethod of the invention, for example, a human or non-human T-cell,preferably a human TCR.

The inventive TCRs, polypeptides, proteins, (including functionalvariants thereof), nucleic acids, recombinant expression vectors, hostcells (including populations thereof), and antibodies (including antigenbinding portions thereof), can be isolated and/or purified. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. For example, the purity can beat least about 50%, can be greater than 60%, 70% o, 80%, 90%, 95%, orcan be 100%.

The inventive antigen recognizing constructs, TCRs, polypeptides,proteins (including functional variants thereof), nucleic acids,recombinant expression vectors, host cells (including populationsthereof), and antibodies (including antigen binding portions thereof),all of which are collectively referred to as “inventive TCR materials”hereinafter, can be formulated into a composition, such as apharmaceutical composition. In this regard, the invention provides apharmaceutical composition comprising any of the antigen recognizingconstructs, TCRs, polypeptides, proteins, functional portions,functional variants, nucleic acids, expression vectors, host cells(including populations thereof), and antibodies (including antigenbinding portions thereof) described herein, and a pharmaceuticallyacceptable carrier, excipient and/or stabilizer. The inventivepharmaceutical compositions containing any of the inventive TCRmaterials can comprise more than one inventive TCR material, e.g., apolypeptide and a nucleic acid, or two or more different TCRs (includingfunctional portions and functional variants thereof). Alternatively, thepharmaceutical composition can comprise an inventive TCR material incombination with another pharmaceutically active agent(s) or drug(s),such as chemotherapeutic agents, e.g., asparaginase, busulfan,carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil,gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab,vinblastine, vincristine, etc. Preferably, the carrier is apharmaceutically acceptable carrier. With respect to pharmaceuticalcompositions, the carrier can be any of those conventionally used forthe particular inventive TCR material under consideration. Suchpharmaceutically acceptable carriers are well-known to those skilled inthe art and are readily available to the public. It is preferred thatthe pharmaceutically acceptable carrier be one, which has no detrimentalside effects or toxicity under the conditions of use.

Thus, also provided is a pharmaceutical composition, comprising any ofthe herein described products of the invention and TCR materials of theinvention, specifically any proteins, nucleic acids or host cells. In apreferred embodiment the pharmaceutical composition is for immunetherapy, preferably adoptive cell therapy.

Preferably, the inventive TCR material is administered by injection,e.g., intravenously. When the inventive TCR material is a host cellexpressing the inventive TCR (or functional variant thereof), thepharmaceutically acceptable carrier for the cells for injection mayinclude any isotonic carrier such as, for example, normal saline (about0.90% w/v of NaCl in water, about 300 mOsm/L NaCl in water, or about 9.0g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott,Chicago, Ill.), PLASMA-LYTE A (Baxter, Deerfield, Ill.), about 5%dextrose in water, or Ringer's lactate. In an embodiment, thepharmaceutically acceptable carrier is supplemented with human serumalbumin.

For purposes of the invention, the amount or dose (e.g., numbers ofcells when the inventive TCR material is one or more cells) of theinventive TCR material administered may be sufficient to affect, e.g., atherapeutic or prophylactic response, in the subject or animal over areasonable time frame. For example, the dose of the inventive TCRmaterial should be sufficient to bind to a cancer antigen, or detect,treat or prevent cancer in a period of from about 2 hours or longer,e.g., 12 to 24 or more hours, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular inventive TCR materialand the condition of the animal (e.g., human), as well as the bodyweight of the animal (e.g., human) to be treated.

It is contemplated that the inventive pharmaceutical compositions, TCRs(including functional variants thereof), polypeptides, proteins, nucleicacids, recombinant expression vectors, host cells, or populations ofcells can be used in methods of treating or preventing cancer, orCOL6A3-positive premalignancy. The inventive TCRs (and functionalvariants thereof) are believed to bind specifically to COL6A3 antigen,such that the TCR (or related inventive polypeptide or protein andfunctional variants thereof), when expressed by a cell, is able tomediate an immune response against a target cell expressing the COL6A3antigens of the invention. In this regard, the invention provides amethod of treating or preventing a condition, in particular cancer, in amammal, comprising administering to the mammal any of the pharmaceuticalcompositions, antigen recognizing constructs, in particular TCRs (andfunctional variants thereof), polypeptides, or proteins describedherein, any nucleic acid or recombinant expression vector comprising anucleotide sequence encoding any of the TCRs (and functional variantsthereof), polypeptides, proteins described herein, or any host cell orpopulation of cells comprising a recombinant vector, which encodes anyof the constructs of the invention (and functional variants thereof),polypeptides, or proteins described herein, in an amount effective totreat or prevent the condition in the mammal, wherein the condition iscancer, preferably COL6A3 positive cancer.

Examples of pharmaceutically acceptable carriers or diluents useful inthe present invention include stabilizers such as SPGA, carbohydrates(e.g., sorbitol, mannitol, starch, sucrose, glucose, dextran), proteinssuch as albumin or casein, protein containing agents such as bovineserum or skimmed milk and buffers (e.g., phosphate buffer).

The terms “treat,” and “prevent” as well as words stemming therefrom, asused herein, do not necessarily imply 100% or complete treatment orprevention. Rather, there are varying degrees of treatment or preventionof which one of ordinary skill in the art recognizes as having apotential benefit or therapeutic effect. In this respect, the inventivemethods can provide any amount of any level of treatment or preventionof a condition in a mammal. Furthermore, the treatment or preventionprovided by the inventive method can include treatment or prevention ofone or more conditions or symptoms of the condition, e.g., cancer, beingtreated or prevented. For example, treatment or prevention can includepromoting the regression of a tumor. Also, for purposes herein,“prevention” can encompass delaying the onset of the condition, or asymptom or condition thereof.

The present invention also relates to a method of treating cancercomprising administering a TCR, a nucleic acid, or a host cell of thepresent description in combination with at least one chemotherapeuticagent and/or radiation therapy.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from said subject;    -   b) transforming the cell with at least one vector encoding an        antigen recognizing construct of the present invention to        produce a transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of treating cancer in a subject in needthereof, comprising:

-   -   a) isolating a cell from a healthy donor;    -   b) transforming the cell with a vector encoding an antigen        recognizing construct of the present invention to produce a        transformed cell;    -   c) expanding the transformed cell to produce a plurality of        transformed cells; and    -   d) administering the plurality of transformed cells to said        subject.

Also provided is a method of detecting cancer in a biological samplecomprising:

-   -   a) contacting the biological sample with an antigen recognizing        construct of the present description;    -   b) detecting binding of the antigen recognizing construct to the        biological sample.

In some embodiments, the method of detecting cancer is carried out invitro, in vivo or in situ.

Also provided is a method of detecting the presence of a condition in amammal. The method comprises (i) contacting a sample comprising one ormore cells from the mammal with any of the inventive TCRs (andfunctional variants thereof), polypeptides, proteins, nucleic acids,recombinant expression vectors, host cells, populations of cells,antibodies, or antigen binding portions thereof, or pharmaceuticalcompositions described herein, thereby forming a complex, and (ii)detecting the complex, wherein detection of the complex is indicative ofthe presence of the condition in the mammal, wherein the condition iscancer, such as a COL6A3-positive malignancy.

With respect to the inventive method of detecting a condition in amammal, the sample of cells can be a sample comprising whole cells,lysates thereof, or a fraction of the whole cell lysates, e.g., anuclear or cytoplasmic fraction, a whole protein fraction, or a nucleicacid fraction.

For purposes of the inventive detecting method, the contacting can takeplace in vitro or in vivo with respect to the mammal. Preferably, thecontacting is in vitro.

Also, detection of the complex can occur through any number of waysknown in the art. For instance, the inventive antigen recognizingconstructs (and functional variants thereof), polypeptides, proteins,nucleic acids, recombinant expression vectors, host cells, populationsof cells, or antibodies or TCRs, or antigen binding portions thereof,described herein, can be labeled with a detectable label such as, forinstance, a radioisotope, a fluorophore (e.g., fluoresceinisothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkalinephosphatase, horseradish peroxidase), and element particles (e.g., goldparticles).

For purposes of the inventive methods, wherein host cells or populationsof cells are administered, the cells can be cells that are allogeneic orautologous to the mammal. Preferably, the cells are autologous to themammal.

With respect to the above mentioned medical applications of the TCRmaterial of the invention, the to be treated and/or diagnosed cancer canbe any cancer, including any of acute lymphocytic cancer, acute myeloidleukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breastcancer, cancer of the anus, anal canal, or anorectum, cancer of the eye,cancer of the intrahepatic bile duct, cancer of the joints, cancer ofthe neck, gallbladder, or pleura, cancer of the nose, nasal cavity, ormiddle ear, cancer of the oral cavity, cancer of the vagina, cancer ofthe vulva, chronic lymphocytic leukemia, chronic myeloid cancer, coloncancer, esophageal cancer, cervical cancer, gastrointestinal carcinoidtumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer,larynx cancer, liver cancer, lung cancer, malignant mesothelioma,melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma,cancer of the oropharynx, ovarian cancer, cancer of the penis,pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynxcancer, prostate cancer, rectal cancer, renal cancer, skin cancer, smallintestine cancer, soft tissue cancer, stomach cancer, testicular cancer,thyroid cancer, cancer of the uterus, ureter cancer, and urinary bladdercancer. A preferred cancer is cancer of the uterine cervix, oropharynx,anus, anal canal, anorectum, vagina, vulva, or penis. A particularlypreferred cancer is a COL6A3 positive cancer, such as gastrointestinalor gastric cancer.

In general, the invention provides a method for treating a subjectsuffering from a tumor or tumor disease comprising the administration ofthe antigen recognizing constructs, nucleic acids, vectors,pharmaceutical compositions and/or host cell as disclosed by the presentinvention. Preferably the subject is a subject in need of such atreatment. The subject in preferred embodiments is a mammalian subject,preferably a human patient, suffering from a tumor or tumor disease,which is COL6A3-positive.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The present invention will now be further described in the followingexamples with reference to the accompanying figures and sequences,nevertheless, without being limited thereto. For the purposes of thepresent invention, all references as cited herein are incorporated byreference in their entireties. In the Figures and Sequences:

FIG. 1 shows the conversion of a TCR into stabilized Vα/Vβ single-chainTCR (scTv) via yeast surface display. ScTv molecules displayed on thesurface of transformed Saccharomyces cerevisiae EBY100 were stained withFITC-labeled anti-Vbeta1 antibody and PE-labeled HLA-A*02/COL6A3-002tetramer. The unmodified scTv R4P3F9 (left panel, SEQ ID NO: 22) iscompared to an scTv clone bearing single point mutations to stabilizethe scTv scaffold (right panel), which was derived from the selection ofa random mutation scTv library.

FIG. 2 shows scTv affinity maturation via yeast surface display.Stabilized scTv molecules with and without affinity maturated CDR1 betawere stained with HLA-A*02 tetramers containing COL6A3-002 (SEQ IDNO: 1) and counterstained with a mix of HLA-A*02 tetramers containing 9peptides (SEQ ID NO: 28 to 36) with high sequence similarity toCOL6A3-002. Stabilized scTv (SEQ ID NO 27) with non-maturated beta chainCDR1 sequence RSGDLS (SEQ ID NO: 13) is compared with scTv clonesbearing the affinity maturated beta chain CDR1 sequences AMDHPY (SEQ IDNO: 40) and ARWHRN (SEQ ID NO: 39).

FIG. 3 shows size exclusion chromatography elution profiles of anti-CD3Fab-scTv R4P3F9S fusion variants 75-1 to 75-25.

FIG. 4 shows the HLA-A*02/COL6A3-002 binding kinetics of anti-CD3Fab-scTv R4P3F9S fusion variants 75-1 to 75-25 as measured by biolayerinterferometry. Analyzed concentrations of HLA-A*02/COL6A3-002 areindicated.

FIG. 5 shows binding analysis of anti-CD3 Fab-scTv R4P3F9S fusionvariants 75-1 to 75-25 as measured via biolayer interferometry (BLI). 1μM of HLA-A*02 in complex with the indicated similar peptides wasanalyzed.

FIG. 6 shows a comparison of HLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001(SEQ ID NO: 30) binding kinetics of different anti-CD3 Fab-scTv R4P3F9Sfusion variants. Analyzed concentrations of Fab-scTv molecules areindicated.

FIG. 7 shows staining of maturated R4P3F9 TCR variant expressing humanCD8⁺ T cells with PE-labeled HLA-A*02/COL6A3-002 tetramers. For controlpurpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 wasexpressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers wasused.

FIG. 8 shows IFN-gamma release of maturated R4P3F9 TCR variantexpressing human CD8⁺ T cells in response to COL6A3-002. For controlpurpose, no TCR (mock) or the 1G4 TCR specific for NYESO1-001 wasexpressed. IFN-gamma release was determined by ELISA after co-culture ofelectroporated CD8⁺ T cells with T2 cells loaded with a serial dilutionof COL6A3-002.

FIG. 9 shows IFN-gamma release of maturated R4P3F9 TCR variantexpressing human CD8⁺ T cells in response to COL6A3-002 and differentsimilar peptides. For control purpose, no TCR (mock) or the 1G4 TCRspecific for NYESO1-001 was expressed. IFN-gamma release was determinedby ELISA after co-culture of electroporated CD8⁺ T cells with T2 cellsloaded with 10 μM of COL6A3-002 or similar peptides.

FIG. 10 shows staining of maturated R4P3F9 TCR variant expressing humanCD8⁺ T cells with PE-labeled peptide-HLA-A*02 tetramers. For controlpurpose, no TCR (Mock) or the 1G4 TCR specific for NYESO1-001 wasexpressed and staining with PE-labeled HLA-A*02/NYESO1-001 tetramers wasused.

FIG. 11 shows IFN-gamma release of maturated R4P3F9 TCR variantexpressing human CD8⁺ T cells in response to COL6A3-002 or COL6A1-001.For control purpose, no TCR (mock) or the 1G4 TCR specific forNYESO1-001 was expressed. IFN-gamma release was determined by ELISAafter co-culture of electroporated CD8⁺ T cells with T2 cells loadedwith a serial dilution of COL6A3-002 or COL6A1-001.

FIG. 12 shows IFN-gamma release of primary human CD8⁺ T cells expressingR4P3F9 TCR variants upon co-culture with different tumor cell lines.SF539, SW982 and Hs840.T cells present the target peptide at differentlevels. MCF-7 cells do not present the target peptide. As controls,effector cells without exogenous TCR were analyzed along with cells withTCRs of interest. IFN-gamma release was determined by ELISA. * marks adata point that is out of scale.

TABLE 1 Peptide sequences of the invention(positions are according to IMGT numbering: (François Ehrenmann, Patrice Duroux, Chantal Ginestoux; Protein displays: human (Homo sapiens) TRAV; IMGT Repertoire. IMGT ® theinternational ImMunoGenetics information system ® w ww.imgt.org.; Created: 16 Mar. 2011. Version: 3 Jun. 2016; François Ehrenmann,Patrice Duroux, Chantal Ginestoux; Protein displays: human (Homo sapiens) TRBV; IMGT Repertoire. IMGT ®, the internationalImMunoGenetics information system ® w ww.imgt.org.;Created: 16 Mar. 2011. Version: 3 Jun. 2016.) SEQ ID NO: NameDescription Sequence  1 COL6A3-002 FLLDGSANV  2 R4P3F9 R4P3F9 TCRMKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha alphaSLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDG chain-RFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ fullLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD lengthFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS  3 R4P3F9 R4P3F9 TCRMKSLRVLLVILWLQLSWVWSQ alpha alpha leader chain- leader peptide  4 R4P3F9R4P3F9 TCR QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS alpha alphaGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variable chain-PSDSATYLCAAYSGAGSYQLTFGKGTKLSVIP variable domain  5 R4P3F9 R4P3F9 TCRDRGSQS CDRa1 alpha chain- CDR1  6 R4P3F9 R4P3F9 TCR IYSNGD CDRa2 alphachain- CDR2  7 R4P3F9 R4P3F9 TCR CAAYSGAGSYQLT CDRa3 alpha chain- CDR3 8 R4P3F9- R4P3F9 TCR NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS alphaalpha DVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS constant chain-IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSSdomain  9 R4P3F9- R4P3F9 TCR NIQN alpha alpha constant chain- startconstant domain start 10 R4P3F9 R4P3F9 TCRMGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta betaRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNIL chain-ERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY fullTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL lengthVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 11R4P3F9 R4P3F9 TCR MGFRLLCCVAFCLLGAGPV beta beta leader chain- leaderpeptide 12 R4P3F9 R4P3F9 TCR DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDbeta beta QGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSS variable chain-LELGDSALYFCASSVESSYGYTFGSGTRLTVV variable domain 13 R4P3F9 R4P3F9 TCRRSGDLS CDRb1 beta chain- CDR1 14 R4P3F9 R4P3F9 TCR YYNGEE CDRb2 betachain- CDR2 15 R4P3F9 R4P3F9 TCR CASSVESSYGYT CDRb3 beta chain- CDR3 16R4P3F9 R4P3F9 TCR EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDH beta betaVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL constant chain-RVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI constantVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYA domain VLVSALVLMAMVKRKDF 17R4P3F9 R4P3F9 TCR EDLNK beta beta chain- constant constant start 1domain start 1 18 R4P3F9 R4P3F9 TCR EDLKN beta beta chain- constantconstant start 2 domain start 2 19 Aga2p- Aga2pMQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL R4P3F9 fusionSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP proteinINTQYVFGGGGSDYKDDDDKGGGASQKEVEQNSGPLSVPE with scTvGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGD R4P3F9 andKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGA tagsGSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLNKAAAG GSGGEQKLISEEDL 20 Aga2p LeaderMQLLRCFSIFSVIASVLAQELTTICEQIPSPTLESTPYSL sequenceSTTTILANGKAMQGVFEYYKSVTFVSNCGSHPSTTSKGSP and Aga2p INTQYVF 21 FLAG tagFLAG tag GGGGSDYKDDDDKGGGAS plus linkers 22 scTv SingleQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9 chainGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ variablePSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG domains ofSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR R4P3F9SGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSA withQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG linker; TRLTVVEDLNK aF55S inalpha variable domain 23 Myc tag Linker and AAAGGSGGEQKLISEEDL Myc tag24 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9- R4P3F9GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bQ43K withPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizingSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutationSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNILERFSA bQ43K inQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variabledomain 25 scTv scTv QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYS R4P3F9-R4P3F9 GKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ bL72S withPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGG stabilizingSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPR mutationSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNISERFSA bL72S inQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSG beta TRLTVVEDLNK variabledomain 26 CDRa1 aG29R DRRSQS mutant1 mutation 27 scTv StabilizedQKEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYS R4P3F9S version ofGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ scTvPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPGGGGSGGG R4P3F9GSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGS GTRLTVV 28 AGRN-001 SimilarALLDGRVQL peptides 29 CLASP1-001 Similar RLLDGAFKL peptides 30COL6A1-001 Similar ILLDGSASV peptides 31 COL6A2-001 Similar FLLDGSERLpeptides 32 COL6A3-006 Similar FLFDGSANLV peptides 33 COL6A3-008 SimilarFLFDGSANL peptides 34 COL6A3-014 Similar FLLDGSEGV peptides 35 VWA2-001Similar FLLDGSNSV peptides 36 VWF-001 Similar FLLDGSSRL peptides 37CDRb1 Beta chain- ARWHNN mutant 1 CDR1 variant 1 38 CDRb1 Beta chain-AKDHLN mutant 2 CDR1 variant 2 39 CDRb1 Beta chain- ARWHRN mutant 3 CDR1variant 3 40 CDRb1 Beta chain- AMDHPY mutant 4 CDR1 variant 4 41 CDRb1Beta chain- ATDHYN mutant 5 CDR1 variant 5 42 CDRb1 Beta chain- ARYHTNmutant 6 CDR1 variant 6 43 CDRb1 Beta chain- APYHLN mutant 7 CDR1variant 7 44 CDRb1 Beta chain- AKDHTN mutant 8 CDR1 variant 8 45 CDRb1Beta chain- ARYHRN mutant 9 CDR1 variant 9 46 CDRb1 Beta chain- ARWHSNmutant 10 CDR1 variant 10 47 CDRb1 Beta chain- ATDHYN mutant 11 CDR1variant 11 48 CDRb1 Beta chain- RWGDLN mutant 12 CDR1 variant 12 49CDRb1 Beta chain- ARDHLN mutant 13 CDR1 variant 13 50 75-1 Fab heavyMKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chain withAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilizedKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTvGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9SGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 51 75- Fab heavyMKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC Fab chainAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ heavyKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY chainGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTSPPSPAPPVAG 52 75- Fab light MKWVTFISLLFLFSSAYSDIQMTQSPSSLSASVGDRVTIT Fab chainCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRF lightSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGT chainKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 53 1G4 alpha 1G4 TCRMETLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVL alphaNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGR chain-LNASLDKSSGRSTLYIAASQPGDSATYLCAVRPTSGGSYI fullPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSSDKSVCLFTD lengthFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 54 1G4 alpha 1G4 TCRMETLLGLLILWLQLQWVSSK leader alpha chain- leader peptide 55 1G4 alpha1G4 TCR QEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPG variable alphaKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQ chain-PGDSATYLCAVRPTSGGSYIPTFGRGTSLIVHP variable domain 56 1G4 alpha 1G4 TCRYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDS constant alphaDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNS chain-IIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGF constant RILLLKVAGFNLLMTLRLWSSdomain 57 1G4 beta 1G4 TCR MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLbeta chain- QCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVP fullNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGE lengthLFFGEGSRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATIL YEILLGKATLYAVLVSALVLMAMVKRKDSRG58 1G4 beta 1G4 TCR MSIGLLCCAALSLLWAGPVNA leader beta chain- leaderpeptide 59 1G4 beta 1G4 TCR GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGvariable beta chain- LRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAA variablePSQTSVYFCASSYVGNTGELFFGEGSRLTVL domain 60 1G4 beta Beta chain-EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDH constant constantVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRL domainRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYA VLVSALVLMAMVKRKDSRG 61NYES01-001 Control SLLMWITQV peptide 62 C-14 C-14; C-5MGFRLLCCVAFCLLGAGPVDSGVTQTPKHLITATGQRVTL beta; C-5 TCR fullRCSPAMDHPYVYWYQQSLDQGLQFLIQYYNGEERAKGNIL beta lengthERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGY beta chainTFGSGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATL with CDRb1VCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPAL mutant 4NDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILY EILLGKATLYAVLVSALVLMAMVKRKDF 63C-14 C-14 TCR MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIA alpha fullSLNCTYSDRRSQSFFWYRQYSGKSPELIMFIYSNGDKEDG lengthRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQ alphaLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTD chain withFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN CDRa1KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETD mutant 1TNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS 64 75-5 Fab heavyMKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chain withAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilizedKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTvGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9SGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS and CDRb1GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant 4KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 65 75-14 Fab heavyMKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chain withAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilizedKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTvGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S,GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS CDRa1GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP mutant 1KSCDKTHTSPPSPAPPVAGQKEVEQNSGPLSVPEGAIASL and CDRb1NCTYSDRRSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRF mutant 4TAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQNGGGGSGGGGSGGGGSGGGGSGGGGSGVTQTPKHLITATGQRVTLRCSPAMDHPYVYWYKQSLDQGLQFLIQYYNGEERAKGNISERFSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKN 66 75-25 Fab heavyMKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSC chain withAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQ stabilizedKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCARSGYY scTvGDSDWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG R4P3F9S inGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS beta/alphaGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP orientation,KSCDKTHTSPPSPAPPVAGGVTQTPKHLITATGQRVTLRC CDRa1SPRSGDLSVYWYKQSLDQGLQFLIQYYNGEERAKGNISER mutant 1FSAQQFPDLHSELNLSSLELGDSALYFCASSVESSYGYTFGSGTRLTVVEDLKNGGGGSGGGGSGGGGSGGGGSGGGGSQKEVEQNSGPLSVPEGAIASLNCTYSDRRSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAAYSGAGSYQLTFGKGTKLSVIPNIQN

EXAMPLES

Native T cell receptors (TCRs) against cancer antigens are often oflower affinity when compared to TCRs targeting viral antigens, and thismay be one possible explanation for tumor immune escape (Aleksic et al.2012). Therefore, it is desirable to have higher affinity TCR variantsdesigned for the use as antigen recognizing constructs in an adoptivecell therapy, or as recognition module of a soluble approach, i.e.,using bispecific molecules (Hickman et al. 2016). This invention thusrelates to the modification and optimization of the naturally occurringT cell receptor R4P3F9 (SEQ ID NOs: 2 and 10) targeting the tumorassociated peptide COL6A3-002 (SEQ ID NO: 1) with an affinity of about60 μM (DE102016115246).

Example 1: Generation of Stable scTv

For the present invention, the previously investigated TCR R4P3F9 (SEQID NOs: 2 and 10) was converted into a single chain TCR construct (scTv,SEQ ID NO: 22) for maturation via yeast surface display by combinationof the variable alpha (SEQ ID NO: 4) and beta (SEQ ID NO: 12) domainwith appendages of the respective constant domains (SEQ ID NOs: 9 and17) and an appropriate glycine-serine linker sequence. The DNA of thecorresponding sequence was synthesized and transformed intoSaccharomyces cerevisiae EB Y100 (MATa AGA1::GAL1AGA1::URA3 ura352 trp1leu2delta200 his3delta200 pep4::HIS3 prbd1.6R can1 GAL) (ATCC® MYA4941™) together with a yeast display vector containing a leader sequenceand the Aga2p yeast mating protein (SEQ ID NO: 20), based on pCT302(Boder et al. 2000). The resulting fusion protein after homologousrecombination in the yeast (SEQ ID NO: 19) contains a leader peptide atthe N-terminus of the Aga2p protein, responsible for the display of theprotein of interest (Boder et al. 1997), short peptide tags includinglinker sequences (SEQ ID NOs: 21 and 23) for expression controls and theprotein of interest, namely the scTv R4P3F9 (SEQ ID NO: 22) or itsvariants. The transformation was performed as described inDE102016121899 and resulted in up to 10⁹ yeast clones per library. Thelibraries were generated via a random mutation PCR approach spanning thewhole gene sequence of the scTv R4P3F9.

The selection process for the yeast clones bearing the best expressingscTv that is selectively binding to COL6A3-002 in context of HLA-A*02was essentially performed as described in Smith et al 2015. In order toascertain high expression and correct conformation of the scTv R4P3F9variant, displayed on the yeast surface, an anti-Vbeta1 (BeckmanCoulter, clone BL37.2) antibody was used, together withHLA-A*02/COL6A3-002 tetramer (FIG. 1). The scTv conversion by yeastsurface display revealed two crucial stabilizing mutations in theframework region together with the original CDR sequences for the properpresentation of the scTv on the cell surface, namely bQ43K (SEQ ID NO:24) and bL72S (SEQ ID NO: 25), both located on the beta chain.Furthermore, during stability maturation position 29 in the CDR1 of thealpha chain (SEQ ID NO: 5) was converted from glycine to arginine (CDRa1mutant 1, SEQ ID NO: 26), which resulted in improved tetramer binding.

Example 2: Affinity Maturation of Stabilized scTv

To generate scTv molecules with higher binding affinity towardsHLA-A*02/COL6A3-002, the CDRb1 (SEQ ID NO: 13) was degenerated using thepreviously identified stabilized scTv R4P3F9S scaffold (SEQ ID NO: 27)expressing the stabilizing mutations aG29R, bQ43K and bL72S. The CDRb1residues were randomized by using degenerate DNA oligo primersessentially as described previously (Smith et al. 2015). The resultingDNA library was transformed as described in example 1. To retain scTvbinding selectivity, negative selection was employed against HLA-A*02tetramers comprising peptides derived from normal tissues (SEQ ID NOs:28 to 36), which show high sequence similarity to COL6A3-002 peptide.

For the selection of affinity enhanced and selective scTv R4P3F9Svariants a decreasing concentration of HLA-A*02/COL6A3-002 tetramer wasused for each sorting round. After three selection rounds, single scTvclones were isolated and sequenced, resulting in a variety of affinitymaturated CDRb1 sequences (SEQ ID NOs: 37 to 49). For scTv withmaturated CDRb1 sequences a strong improvement in COL6A3-002 bindingcould be demonstrated while the selectivity of COL6A3-002 binding wasretained as no binding of 9 similar peptides was observed (FIG. 2).

Example 3: Production of Bispecific Antibody-scTv Fusion Proteins

Stabilized and affinity maturated scTv against HLA-A*02/COL6A3-002 canbe expressed in fusion with an antibody moiety directed against CD3allowing tumor-specific retargeting and activation of T cellsindependent of their natural specificity. The inventors generatedbispecific antibody-TCR fusion proteins comprising an anti-CD3 Fab(UCHT1) heavy chain (SEQ ID NO: 51) fused to scTv R4P3F9S variants (SEQID NOs: 50, 64, 65 and 66) and an anti-CD3 Fab (UCHT1) light chain (SEQID NO: 52). The resulting Fab-scTv fusion proteins have a molecular massof approximately 75 kDa. Based on different CDR1 sequences of the scTvR4P3F9S alpha (SEQ ID NOs: 5 and 26) and beta chain (SEQ ID NOs: 13 and37 to 49) different Fab-scTv fusion variants (75-1 to 75-25, Table 2)were expressed in transiently transfected ExpiCHO cells as recommendedby the manufacturer. Proteins were purified by protein L and sizeexclusion chromatography. All fusion variants could be produced withyields ranging from 80 μg up to 1 mg (Table 2) and homogeneously formedheterodimers at the expected size as analyzed by size exclusionchromatography (FIG. 3).

TABLE 2 Nomenclature and yields of bispecificFab-scTv fusion proteins. The molecules arebased on SEQ ID NOs 50 and 52 and theindicated CDRa1 and CDRb1 variants. Yield Variant CDRa1/SEQ CDRb1/SEQ[μg] 75-1 DRGSQS RSGDLS  267.9 (SEQ ID NO. 5) (SEQ ID NO. 13) 75-2DRGSQS ARWHNN   78.4 (SEQ ID NO. 5) (SEQ ID NO. 37) 75-3 DRGSQS AKDHLN 646.7 (SEQ ID NO. 5) (SEQ ID NO. 38) 75-4 DRGSQS ARWHRN  704.3(SEQ ID NO. 5) (SEQ ID NO. 39) 75-5 DRGSQS AMDHPY  397.2 (SEQ ID NO. 5)(SEQ ID NO. 40) 75-6 DRGSQS ATDHYN  268.1 (SEQ ID NO. 5) (SEQ ID NO. 41)75-7 DRGSQS ARYHTN   83.2 (SEQ ID NO. 5) (SEQ ID NO. 42) 75-8 DRGSQSAPYHLN  765.7 (SEQ ID NO. 5) (SEQ ID NO. 43) 75-9 DRGSQS AKDHTN 1067.2(SEQ ID NO. 5) (SEQ ID NO. 44) 75-10 DRRSQS RSGDLS  389.6(SEQ ID NO. 26) (SEQ ID NO. 13) 75-11 DRRSQS ARWHNN  270.4(SEQ ID NO. 26) (SEQ ID NO. 37) 75-12 DRRSQS AKDHLN  943.6(SEQ ID NO. 26) (SEQ ID NO. 38) 75-13 DRRSQS ARWHRN  560.3(SEQ ID NO. 26) (SEQ ID NO. 39) 75-14 DRRSQS AMDHPY  360.7(SEQ ID NO. 26) (SEQ ID NO. 40) 75-15 DRRSQS ATDHYN  541.5(SEQ ID NO. 26) (SEQ ID NO. 41) 75-16 DRRSQS ARYHTN  403.6(SEQ ID NO. 26) (SEQ ID NO. 42) 75-17 DRRSQS APYHLN  195.5(SEQ ID NO. 26) (SEQ ID NO. 43) 75-18 DRRSQS AKDHTN  731.3(SEQ ID NO. 26) (SEQ ID NO. 44) 75-19 DRRSQS ARYHRN  794  (SEQ ID NO. 26) (SEQ ID NO. 45) 75-20 DRRSQS ARWHSN   85.5(SEQ ID NO. 26) (SEQ ID NO. 46) 75-21 DRRSQS ATDHYN  276  (SEQ ID NO. 26) (SEQ ID NO. 47) 75-22 DRRSQS RWGDLN  255  (SEQ ID NO. 26) (SEQ ID NO. 48) 75-23 DRRSQS ARDHLN  217  (SEQ ID NO. 26) (SEQ ID NO. 49) 75-24^(a) DRGSQS RSGDLS  166.6(SEQ ID NO: 5) (SEQ ID NO. 13) 75-25^(a) DRRSQS RSGDLS  267  (SEQ ID NO. 26) (SEQ ID NO. 13) ^(a)beta-alpha orientation of scTv

Example 4: Fab-scTv Fusion Protein Binding to COL6A3-002 and SimilarPeptides

Binding affinity of anti-CD3-scTv R4P3F9S fusion proteins towardsHLA-A*02 monomers with COL6A3-002 or different similar peptides wasmeasured by biolayer interferometry. Measurements were done on an OctetRED384 system using settings recommended by the manufacturer. Briefly,purified Fab-scTv molecules were loaded onto biosensors (FAB2G) prior toanalyzing serial dilutions of HLA-A*02/COL6A3-002. Compared to variants75-1 and 75-24 comprising wild-type CDRa1 and wild-type CDRb1 increasedbinding affinities of up to 40-fold were observed for Fab-scTv variantswith maturated CDRa1 and/or CDRb1 sequences (Table 3, FIG. 4). In orderto evaluate the selectivity of binding to HLA-A*02/COL6A3-002, purifiedFab-scTv molecules loaded onto FAB2G biosensors were screened forbinding to 1 μM similar peptides (SEQ ID NOs: 28 to 36), each in complexwith HLA-A*02. Except of HLA-A*02/COL6A1-001 (SEQ ID NO: 30), which wasbound by most of the Fab-scTv variants containing maturated CDRa1 (SEQID NO: 26), Fab-scTv variants showed no binding to similar peptides(FIG. 5) arguing for high binding selectivity. For some Fab-scTvvariants the therapeutic window between HLA-A*02/COL6A3-002 andHLA-A*02/COL6A1-001 binding was investigated by loading biotinylatedpeptide-HLA complexes onto biosensors (SA) and analyzing dilution seriesof Fab-scTv variants. While variant 75-10 comprising maturated CDRa1(SEQ ID NO: 26) and wild-type CDRb1 (SEQ ID NO: 13) sequence showed8-fold increased binding affinity to HLA-A*02/COL6A3-002 overHLA-A*02/COL6A1-001, an up to 57-fold increased binding affinity wasdetected for Fab-scTv variant 75-13 comprising a maturated CDRb1 (SEQ IDNO: 39) arguing for an improvement in therapeutic window (Table 4, FIG.6).

TABLE 3 Binding affinity of Fab-scTv fusion proteins toHLA-A*02/COL6A3-002. Variant KD (M) kon (1/Ms) koff (1/s) 75-1 8.06E−061.01E+05 8.17E−01 75-2 3.69E−06 1.59E+05 5.86E−01 75-3 4.92E−06 9.71E+044.78E−01 75-4 5.76E−06 9.78E+04 5.63E−01 75-5 4.32E−04 2.21E+03 9.55E−0175-6 1.13E−06 2.06E+05 2.32E−01 75-7 1.79E−06 1.93E+05 3.44E−01 75-83.45E−06 1.36E+05 4.69E−01 75-9 1.41E−05 6.02E+04 8.51E−01 75-101.78E−06 1.69E+05 3.01E−01 75-11 2.82E−07 4.16E+05 1.18E−01 75-123.74E−07 2.67E+05 1.00E−01 75-13 4.05E−07 3.28E+05 1.33E−O1 75-143.10E−06 8.41E+04 2.61E−01 75-15 7.78E−07 2.33E+05 1.81E−01 75-165.87E−07 3.37E+05 1.98E−01 75-17 2.27E−07 3.62E+05 8.20E−02 75-181.93E−06 1.51E+05 2.91E−01 75-19 6.00E−07 2.96E+05 1.78E−01 75-205.31E−07 6.08E+05 3.23E−01 75-21 5.52E−07 2.72E+05 1.50E−01 75-228.22E−07 2.48E+05 2.04E−01 75-23 3.24E−07 3.18E+05 1.03E−01 75-245.20E−06 1.08E+05 5.62E−01 75-25 8.33E−06 6.23E+04 5.19E−01

TABLE 4 Comparative binding affinity of Fab-scTv fusion proteins toHLA-A*02/COL6A3-002 and HLA-A*02/COL6A1-001. Variant pHLA-A*02 KD (M)KD_(COL6A1-001)/KD_(COL6A3-002) 75-10 COL6A3-002 1.37E−05 8 COL6A1-0011.08E−04 75-11 COL6A3-002 8.50E−07 8 COL6A1-001 6.46E−06 75-12COL6A3-002 7.24E−07 12 COL6A1-001 8.98E−06 75-13 COL6A3-002 7.39E−07 57COL6A1-001 4.23E−05 75-17 COL6A3-002 8.25E−07 9 COL6A1-001 7.10E−0675-23 COL6A3-002 1.15E−06 22 COL6A1-001 2.55E−05

Example 5: Use of Affinity-Maturated TCRs for Cellular Expression

Modification of T cells to express TCRs recognizing a tumor-specificpeptide-HLA is a promising alternative of redirecting T cells to cancercells. As the usage of maturated CDR1 sequences could improve cell-boundTCRs against HLA-A*02/COL6A3-002, the identified CDRa1 and CDRb1 mutantsequences were grafted onto the parental TCR R4P3F9 (SEQ ID NOs: 2 and10). The resulting mutant TCR variants (C-1 to C-18, Table 5) wereexpressed in human CD8⁺ T cells after electroporation of respective mRNAgenerated by in vitro transcription of PCR-amplified DNA constructs. Forcontrol purpose, the 1G4 TCR (SEQ ID NOs: 53 and 57) against NYESO1-001peptide (SEQ ID NO: 61) was expressed. After overnight incubation ofRNA-electroporated CD8⁺ T cells, expression of introduced TCR variantswas analyzed by staining with PE-labeled HLA-A*02/COL6A3-002 tetramersor HLA-A*02/NYESO1-001 tetramers. While the parental TCR R4P3F9 variantC-1 showed only minimal staining with HLA-A*02/COL6A3-002 tetramers, theR4P3F9 TCR variants C-2 to C-18 with maturated CDRa1 and/or CDRb1 showedincreased tetramer staining (FIG. 7). Functional activation of CD8⁺ Tcells (20,000 cells/well) expressing different maturated R4P3F9 TCRvariants was investigated by determining levels of released IFN-gammaupon co-culture with T2 cells (20,000 cells/well) loaded with either adilution series of COL6A3-002 (SEQ ID NO: 1) or 10 μM of COL6A3-002 andsimilar peptides (SEQ ID NOs: 28 to 36). Compared to the parental R4P3F9TCR variant C-1, maturated TCR variants C-2 to C-18 showed increasedIFN-gamma release with maximum levels reached already at lower peptideconcentrations (FIG. 8). As expected no IFN-gamma release was observedwith T cells expressing no TCR or the 1G4 control TCR specific forNYESO1-001. To analyze the selectivity of COL6A3-002 recognition of thematurated R4P3F9 TCR variants, the IFN-gamma release in response to T2cells loaded with different similar peptides (SEQ ID NOs: 28 to 36) wasanalyzed and revealed different selectivity profiles for the maturatedR4P3F9 TCR variants. Most interestingly, the TCR variants C-5 (SEQ IDNos 62 and 2) and C-14 (SEQ ID NOs: 62 and 63) comprising the samematurated CDRb1 (SEQ ID NO: 40) did not show any cross-reactivitytowards COL6A1-001 or other similar peptides (FIG. 9) making affinitymaturated R4P3F9 TCR variants C-5 and C14 most promising candidates forcellular TCR-based tumor targeting.

TABLE 5 Nomenclature of cellular TCR variants. The molecules are basedon SEQ ID NOs 2 and 10 and the indicated CDRa1 and CDRb1 variants.Variant CDRa1 CDRb1 C-1 DRGSQS (SEQ ID NO. 5) RSGDLS (SEQ ID NO. 13) C-2DRGSQS (SEQ ID NO. 5) ARWHNN (SEQ ID NO. 37) C-3 DRGSQS (SEQ ID NO. 5)AKDHLN (SEQ ID NO. 38) C-4 DRGSQS (SEQ ID NO. 5) ARWHRN (SEQ ID NO. 39)C-5 DRGSQS (SEQ ID NO. 5) AMDHPY (SEQ ID NO. 40) C-6 DRGSQS (SEQ ID NO.5) ATDHYN (SEQ ID NO. 41) C-7 DRGSQS (SEQ ID NO. 5) ARYHTN (SEQ ID NO.42) C-8 DRGSQS (SEQ ID NO. 5) APYHLN (SEQ ID NO. 43) C-9 DRGSQS (SEQ IDNO. 5) AKDHTN (SEQ ID NO. 44) C-10 DRRSQS (SEQ ID NO. 26) RSGDLS (SEQ IDNO. 13) C-11 DRRSQS (SEQ ID NO. 26) ARWHNN (SEQ ID NO. 37) C-12 DRRSQS(SEQ ID NO. 26) AKDHLN (SEQ ID NO. 38) C-13 DRRSQS (SEQ ID NO. 26)ARWHRN (SEQ ID NO. 39) C-14 DRRSQS (SEQ ID NO. 26) AMDHPY (SEQ ID NO.40) C-15 DRRSQS (SEQ ID NO. 26) ATDHYN (SEQ ID NO. 41) C-16 DRRSQS (SEQID NO. 26) ARYHTN (SEQ ID NO. 42) C-17 DRRSQS (SEQ ID NO. 26) APYHLN(SEQ ID NO. 43) C-18 DRRSQS (SEQ ID NO. 26) AKDHTN (SEQ ID NO. 44)

Example 6: Window of COL6A3-002 and COL6A1-001 Recognition of CellularTCR Variants

Cellular expression and analysis of R4P3F9 variants was performed asdescribed above. In accordance with previous experiments (FIG. 7),staining of T cells expressing R4P3F9 TCR variants C-2 to C-18 withPE-labeled HLA-A*02/COL6A3-002 tetramers were increased compared to theparental TCR C-1. Additionally, TCR variants C-12 and C-17 showedbinding to HLA-A*02/COL6A1-001 (FIG. 10). Expression of all maturatedR4P3F9 variants improved functional activation of CD8⁺ T cells inresponse to T2 cells loaded with a dilution series of COL6A3-002 (SEQ IDNO: 1) reaching 5- to 90-fold lower EC₅₀ values compared to the parentalTCR C-1 (FIG. 11, Table 6). The lowest EC₅₀ value was found for variantC-14. Again, TCR variants C-5 (SEQ ID NOs 62 and 2) and C-14 (SEQ IDNOs: 62 and 63) comprising the same maturated CDRb1 (SEQ ID NO: 40) didnot show any cross-reactivity towards COL6A1-001, while other variantsshowed strong recognition with EC₅₀ windows (COL6A3-002 vs. COL6A1-001)as low as factor 5.

TABLE 6 EC₅₀ values [nM] of IFN-γ release of T cells expressing R4P3F9variants after coculture with T2 cells loaded with COL6A3-002 orCOL6A1-001. Variant EC₅₀ COL6A3-002 [nM] EC₅₀ COL6A1-001 [nM] C-1 2.51 —C-2 0.16 — C-3 0.14 871^(a)   C-4 0.13 — C-5 0.15 — C-6 0.48 — C-7 0.29— C-8 0.20 350    C-9 0.55 — C-10 0.32 1.5 C-11 0.32 8.2 C-12 0.20 1.9C-13 0.23 9.7 C-14 0.03 — C-15 0.31 69   C-16 0.34 78   C-17 0.33 4.1C-18 0.14 280089^(a)    ^(a)plateau not reached

Example 7: Efficacy of Maturated R4P3F9 Variants C-5 and C-14 on TumorCell Lines

Cellular expression and analysis of R4P3F9 variants was performed asdescribed above.

Expression of the maturated R4P3F9 variants C-5 (SEQ ID NOs 62 and 2)and C-14 (SEQ ID NOs: 62 and 63) improved functional activation of CD8⁺T cells in response to COL6A3-002 (SEQ ID NO: 1)-presenting tumor celllines as compared to the parental TCR C-1 (FIG. 12). The tumor celllines used during this study present different amounts of targetpeptide. SF539 cells carry ˜4000 copies of HLA-A*02/COL6A3-002 per celland SW982 cells carry ˜460 copies per cell. Whereas the parental TCR C-1did not mediate strong T-cell activation upon co-culture withtarget-positive cell lines, TCR variant C-14 showed even strongerimprovement of functional activation than TCR variant C-5. These dataare in line with EC₅₀ improvements from TCR C-1 to C-5 and to C-14(table 6). The target-negative tumor cell line MCF-7 was not recognizedby any of these TCRs.

REFERENCES

-   Aleksic et al. 2012: Different affinity windows for virus and    cancer-specific T-cell receptors—implications for therapeutic    strategies, Eur J Immunol. 2012 December; 42(12):3174-9;-   Hickman et al. 2016: Antigen Selection for Enhanced Affinity T-Cell    Receptor-Based Cancer Therapies, J Biomol Screen. 2016 September;    21(8):769-85;-   Boder and Wittrup 2000: Yeast surface display for directed evolution    of protein expression, affinity, and stability, Methods Enzymol.    2000; 328:430-44;-   Boder and Wittrup 1997: Yeast surface display for screening    combinatorial polypeptide libraries, Nat Biotechnol. 1997 June;    15(6):553-7;-   Smith et al. 2015: T Cell Receptor Engineering and Analysis Using    the Yeast Display Platform, Methods Mol Biol. 2015; 1319:95-141;-   DE102016121899.5-   DE102016115246

The invention claimed is:
 1. A method of treating a patient who hascancer that presents a peptide consisting of the amino acid sequence ofFLLDGSANV (SEQ ID NO: 1) in a complex with HLA-A*02, comprisingadministering to the patient a population of transformed T cellsexpressing at least one vector encoding a T cell receptor (TCR), whereinthe TCR comprises SEQ ID NOs: 26, 6, 7, 40, 14, and 15, wherein the TCRbinds to a peptide consisting of the amino acid sequence of FLLDGSANV(SEQ ID NO: 1) in a complex with HLA-A*02, and wherein the cancer isselected from breast cancer, gallbladder cancer, colon cancer,esophageal cancer, gastrointestinal carcinoid tumor, lung cancer,melanoma, non-Hodgkin lymphoma, pancreatic cancer, rectal cancer,gastric cancer, and urinary bladder cancer.
 2. The method of claim 1,wherein the population of transformed cells are produced by a methodcomprising isolating a T cell from a subject, transforming the cell withat least one vector encoding the TCR to produce a transformed cell, andexpanding the transformed cell to produce the population of transformedcells.
 3. The method of claim 2, wherein the subject is the patient. 4.The method of claim 1, wherein said T cells are CD8+ T cells.
 5. Themethod of claim 1, wherein the TCR comprises an α chain comprising anamino acid sequence with at least 95% identity to SEQ ID NO: 63 and a βchain comprising an amino acid sequence with at least 95% identity toSEQ ID NO:
 62. 6. The method of claim 1, wherein the population oftransformed cells are administered in the form of a pharmaceuticalcomposition.
 7. The method of claim 6, wherein the pharmaceuticalcomposition comprises a chemotherapeutic agent selected from the groupconsisting of asparaginase, busulfan, carboplatin, cisplatin,daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,methotrexate, paclitaxel, rituximab, vinblastine, and vincristine. 8.The method of claim 1, wherein the TCR comprises: a CDR1α chaincomprising the amino acid sequence of SEQ ID NO: 26 a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 6, a CDR3α chaincomprising the amino acid sequence of SEQ ID NO: 7, a CDR1β chaincomprising the amino acid sequence of SEQ ID NO: 40, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 14, and a CDR3β chaincomprising the amino acid sequence of SEQ ID NO:
 15. 9. The method ofclaim 1, wherein the TCR comprises: a CDR1α chain consisting of theamino acid sequence of SEQ ID NO: 26 a CDR2α chain comprising the aminoacid sequence of SEQ ID NO: 6, a CDR3α chain comprising the amino acidsequence of SEQ ID NO: 7, a CDR1β chain consisting of the amino acidsequence of SEQ ID NO: 40, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 14, and a CDR3β chain comprising the amino acidsequence of SEQ ID NO:
 15. 10. The method of claim 1, wherein the TCRcomprises: a CDR1α chain comprising the amino acid sequence of SEQ IDNO: 26 a CDR2α chain consisting of the amino acid sequence of SEQ ID NO:6, a CDR3α chain comprising the amino acid sequence of SEQ ID NO: 7, aCDR1β chain comprising the amino acid sequence of SEQ ID NO: 40, a CDR2βchain consisting of the amino acid sequence of SEQ ID NO: 14, and aCDR3β chain comprising the amino acid sequence of SEQ ID NO:
 15. 11. Themethod of claim 1, wherein the TCR comprises: a CDR1α chain comprisingthe amino acid sequence of SEQ ID NO: 26 a CDR2α chain comprising theamino acid sequence of SEQ ID NO: 6, a CDR3α chain consisting of theamino acid sequence of SEQ ID NO: 7, a CDR1β chain comprising the aminoacid sequence of SEQ ID NO: 40, a CDR2β chain comprising the amino acidsequence of SEQ ID NO: 14, and a CDR3β chain consisting of the aminoacid sequence of SEQ ID NO:
 15. 12. The method of claim 1, wherein theTCR comprises: a CDR1α chain consisting of the amino acid sequence ofSEQ ID NO: 26 a CDR2α chain consisting of the amino acid sequence of SEQID NO: 6, a CDR3α chain comprising the amino acid sequence of SEQ ID NO:7, a CDR1β chain comprising the amino acid sequence of SEQ ID NO: 40, aCDR2β chain consisting of the amino acid sequence of SEQ ID NO: 14, anda CDR3β chain comprising the amino acid sequence of SEQ ID NO:
 15. 13.The method of claim 1, wherein the TCR comprises: a CDR1α chainconsisting of the amino acid sequence of SEQ ID NO: 26 a CDR2α chaincomprising the amino acid sequence of SEQ ID NO: 6, a CDR3α chainconsisting of the amino acid sequence of SEQ ID NO: 7, a CDR1β chainconsisting of the amino acid sequence of SEQ ID NO: 40, a CDR2β chaincomprising the amino acid sequence of SEQ ID NO: 14, and a CDR3β chainconsisting of the amino acid sequence of SEQ ID NO:
 15. 14. The methodof claim 1, wherein the TCR comprises: a CDR1α chain comprising theamino acid sequence of SEQ ID NO: 26 a CDR2α chain consisting of theamino acid sequence of SEQ ID NO: 6, a CDR3α chain consisting of theamino acid sequence of SEQ ID NO: 7, a CDR1β chain comprising the aminoacid sequence of SEQ ID NO: 40, a CDR2β chain consisting of the aminoacid sequence of SEQ ID NO: 14, and a CDR3β chain consisting of theamino acid sequence of SEQ ID NO:
 15. 15. The method of claim 1, whereinthe TCR comprises: a CDR1α chain consisting of the amino acid sequenceof SEQ ID NO: 26 a CDR2α chain consisting of the amino acid sequence ofSEQ ID NO: 6, a CDR3α chain consisting of the amino acid sequence of SEQID NO: 7, a CDR1β chain consisting of the amino acid sequence of SEQ IDNO: 40, a CDR2β chain consisting of the amino acid sequence of SEQ IDNO: 14, and a CDR3β chain consisting of the amino acid sequence of SEQID NO:
 15. 16. The method of claim 1, wherein the cancer is lung cancer.17. The method of claim 1, wherein the cancer is breast cancer.
 18. Themethod of claim 1, wherein the cancer is non-Hodgkin lymphoma.
 19. Themethod of claim 2, wherein the subject is a healthy donor.
 20. Themethod of claim 1, wherein the T cells are CD4+ T cells.